Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

In order to assess the bioenergetics of blood cells, whole blood is collected from human subjects by venipuncture in an EDTA or ACD vacutainer collection tube. The isolation of leukocytes and platelets following blood collection is visualized in Figure 1A as detailed in the protocol. Whole blood samples are processed within 8 hr of collection to avoid cell death and activation. Extended storage times greater than 8 hr have not been tested but may result in altered bioenergetics and be less representative of physiological conditions. Acid citrate dextrose (ACD-8 ml) and EDTA vacutainer tubes have been used for cell isolations with no observed effect on the bioenergetic function of isolated cells. Anticoagulants are necessary as clots reduce the number of cells obtained, interfere with proper phase formation during Ficoll gradient centrifugation, and result in little to no platelets obtained in centrifuged serum. All blood represents a biological hazard and proper personal protective equipment must be implemented throughout the entire procedure even after cell populations are isolated or cell lysates are generated. Samples are to be disposed of in accordance with the institution’s human waste disposal standards.

Whole blood is centrifuged to separate the platelet rich plasma from the buffy coat, the white layer just above the packed red blood cells which contains the leukocytes. The buffy coat is applied to density gradients and centrifuged to obtain the peripheral blood mononuclear cells (monocytes and lymphocytes) and polymorphonuclear cells (granulocytes). RBC contamination of the individual MNC and PMN cell phases is common at this stage and should be resolved during the MACS purification. The various cell types are then obtained by incubation with magnetic antibodies to obtain pure fractions. Monocytes are collected by incubation of the PBMC fraction with CD14, the flow-through from the monocytes contain lymphocytes, which are then depleted of RBCs and platelets. The neutrophils are obtained by incubation of the polymorphonuclear cell layer with CD15 antibody (Figure 1A). Thereafter, the platelets can be pelleted by centrifugation of the platelet rich plasma and washed to remove other plasma components. After these selections the platelets, CD14+ monocytes, lymphocytes, CD15+ neutrophils can be counted and plated on a cell adhesion medium-coated XF analyzer plate for bioenergetics and oxidative burst analysis.

Each stage of the procedure should be performed in sterile conditions and is encouraged to prevent the premature activation of the oxidative burst response in monocytes and neutrophils. An indicator of less than sterile conditions during the isolation is evident when neutrophils, which have little to no oxygen consumption under basal conditions, begin the extracellular flux assay with elevated OCR measurements that gradually increase or decline throughout the assay. We stress the importance of imaging cells using light microscopy at 200X or greater to ensure that no morphological signs of activation have occurred prior to the assay. For example, neutrophils normally display a rounded to irregular shape with pronounced cell boundaries as seen in Figure 1B. However, these cells will flatten against the surface of the plate when activated and lose their distinct cell morphology. We have previously reported the morphological changes of activated leukocytes and platelets using this protocol and additional measures must be taken to ensure sterile conditions if encountering activation⁸.

A simplified scheme of the protocol is seen in Figure 2 as a time table in which the primary steps of the cell isolation, XF plate preparation, and XF assay are detailed. Isolation consists of the density gradient separation of cell types and is followed by magnetic separation. Parallel to the cell isolation process, XF plate preparation is necessary to avoid delays in cell plating or beginning the XF assay. This is critical because significant delays can lead to cell activation and diminished bioenergetics parameters.

Inadequate cell concentrations after isolation are a common problem and optimization of each centrifugation process is necessary to avoid cell loss. Successful isolations have been performed on as little as 6 ml of blood but alterations can be made depending on circulating concentrations of the desired cell type. If the density gradient is improperly prepared, distinct cell phases might not be visible and loss of cells can occur (see JoVE instructional video for a visual demonstration). Insufficient platelet isolated from the PRP is rare but has occurred if washing buffer does not contain PGI₂ or if the isolation takes place below room temperature. The risk of platelet activation is often prevented if platelets are the last cells to be isolated; however, if platelets remain in washing buffer for extended periods the bioenergetics will be affected. There have been instances of smaller platelet populations that did not pellet during the 1,500 x g centrifugation and the bioenergetics of these cells has not been extensively characterized. See Table 2 for a more complete troubleshooting guide.

The bioenergetic profiles of leukocytes and platelets can be determined using the XF analyzer, which measures real-time O₂ consumption in cells noninvasively. Each cell type is plated on the XF24 plate with five replicates as shown in the Figure 3A. Table 1 indicates the expected basal and oxidative burst values by cell type during the assay and the average protein concentrations per well. Higher throughput technologies are available, such as the XF96, which allows for four donors to be assessed simultaneously as shown in Figure 3B. After the assay, the data is exported to a work-station computer for analysis using the appropriate XF software and Microsoft excel. The OCR values were normalized to total protein content in the corresponding wells and expressed as pmol/min/mg protein. The representative bioenergetic-oxidative burst profiles of each cell type were recently reported by our group⁸. Further analysis of bioenergetic parameters of the assay (basal, ATP-linked, proton leak, maximal, nonmitochondrial and oxidative burst) can be calculated for individual wells as previously shown and described below⁸.

The basal oxygen consumption rate (OCR) is established by the first 3 measurements as shown in Figure 4A. Oligomycin (0.5 µg/ml), an inhibitor of mitochondrial ATP-synthase is injected into the XF medium to estimate the OCR coupled to ATP synthesis and represented as ATP-linked. The residual OCR minus the nonmitochondrial OCR can be attributed to proton leak. Then FCCP an uncoupler is added to determine the maximal OCR, followed by antimycin-A, an inhibitor of mitochondrial respiration, to determine nonmitochondrial sources of oxygen consumption. Reserve capacity is a measure of the amount of ATP that can be produced under energetic demand and can be calculated as the difference between the maximum rate of respiration and the basal. In order to determine the oxidative burst capacity, phorbol 12-myristate 13-acetate (PMA) a PKC agonist is injected to increase NADPH oxidase activity, and the increase in oxygen consumption rate following PMA stimulation can be measured using the XF analyzer. Figure 4B show how to calculate the different parameters of mitochondrial function and oxidative burst.

Figure 1. Isolation of Leukocytes and Platelets from Whole Blood. A) Freshly collected specimens are separated into their plasma and cellular components by centrifugation and purified by Ficoll density gradient, and Magnetic Activated Cell Sorting (MACS) by positive (monocyte and neutrophil) or negative selection (lymphocytes), while platelets are isolated by high-speed centrifugation of the platelet rich plasma. B) Images of the isolated cell populations once resuspended in XF DMEM and plated at indicated cell densities. Please click here to view a larger version of this figure.

Figure 2. Preparation of XF24 plates for Bioenergetic studies of leukocytes and platelets. Detailed timeline for the preparation of a XF24 assay plate by cell isolation and plating and cartridge hydration and injection port loading.

Figure 3. Layout of leukocytes and platelets on XF24 and XF96 analyzer plates. A) Leukocytes (125-250k cells/well) and platelets (25 x 10⁶ cells/well) from a single donor are plated on the XF24 assay plate among background controls. 75 µl of 10x stocks of oligomycin (0.5 µg / ml), FCCP (0.6 µM), antimycin-A (10 µM), and PMA (100 ng/ml) diluted in XF media are loaded into the indicated injection ports. B) Leukocytes (75-150k cells/well) and platelets (10 x10⁶ cells/well) from as many as four donors can be plated on the XF96 in a total volume of 180 µl XF-DMEM. 20 µl of 10x stocks of oligomycin (1.0 µg/ml), FCCP (0.6 µM), antimycin-A (10 µM), and PMA (100 ng/ml) diluted in XF-DMEM are loaded into the indicated injection ports.

Figure 4. Analysis of Bioenergetic indices. A) Representative trace of oxygen consumption rate (OCR) by mitochondria and oxidative burst in monocytes. Basal OCR is established (first 3 rates indicated by numbers in circles). Then sequential additions of oligomycin (O; 0.5 µg/ml), FCCP (F; 0.6 µM) and antimycin-A (A; 10 µM) are injected to determine mitochondrial and nonmitochondrial respiration of nonactivated cells. Finally, PMA (100 ng/ml) is added to measure oxidative burst. B) Modular analysis of mitochondrial function and oxidative burst are calculated by the differences in the rates as indicated.

Table 1. Normal parameters for the XF24 assay: The average basal (Rate 3) and oxidative burst (Rate 7) OCR not corrected to nonmitochondrial OCR or protein are shown by cell type plated at the indicated cell densities. The average protein content per well is shown as performed by a DC Lowry assay. These data represent the mean values of 6-8 healthy donors with parenthesis containing ± SEM.

Table 2. Leukocyte and platelet isolation troubleshooting. The table lists common problems encountered during leukocyte and platelet cell isolation from whole blood and solutions that may guide users through the troubleshooting process.